Ethanol is the most important renewable fuel in terms of volume and market value. Today, the so called first-generation bioethanol is primarily produced from the fermentation of sugars deriving form food crops. This has raised several ethical, environmental and political concerns. Second-generation biofuels can be produced using waste lignocellulosic biomass such as agricultural and forestry residues, or dedicated crops grown on marginal lands. Lignocelluloses are composed of cellulose, hemicellulose, lignin, and several inorganic materials. The process for producing ethanol and other biofuels from lignocellulosic biomass includes: biomass pretreatment, enzymatic hydrolysis and detoxyfication, fermentation and distillation-rectificationdehydration. In this chapter we describe advancements in ethanol production from lignocellulosic biomass with a specific focus on the selection and development of the biocatalysts used in the hydrolysis and fermentation steps.